Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene

Shahhoseini, Maryam; Ziaee, Abed-Ali; Ghaemi, Nasser

doi:10.1046/j.1365-2672.2003.02082.x

Cited by 25 publications

(15 citation statements)

References 18 publications

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“…The pelB signal peptide was found from Erwinia and shown to have the capability of directing the synthesized foreign protein through the cytoplasmic membrane of E. coli [23]. Signal peptide from Bacillus was also successfully used for expression and secretion of a-amylase in E. coli [14], which agrees with our result that the signal peptide from B. subtilis has the function of helping secretion of heterologous protein in E. coli. The comparison between the secretion efficiency promoted by PelB and the nattokinase signal peptides needs further study.…”

Section: Discussionsupporting

confidence: 87%

“…Though the production (49.3 mg l -1 ) of active nattokinase is not comparative with that of B. subtilis, its short growth cycle, simple cultural medium and the easy purification technique make this strategy a potential way in producing this enzyme. For there are reports of high production of proteins in E. coli by secretion means [13,14], production of active nattokinase in E. coli could also be further enhanced by improving secretion efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…Human growth hormone and alpha-amylase were successfully produced in E. coli by the secretion means [13,14]. It seems that expression of fibrinolytic enzyme in E. coli by secretory method is a feasible way to produce active enzyme.…”

Section: Introductionmentioning

confidence: 99%

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Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli

et al. 2007

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Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l(-1) isopropyl-beta-D- thiogalactopyranoside (IPTG) at 20 degrees C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

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Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli

et al. 2007

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“…In most cases, the enzyme is produced extracellularly and recovered from the cultivation medium or periplasmic space (26,29). While some reports have described the intracellular production of ␣-amylase in E. coli (17,23), extracellular production is generally preferred in the industrial context to facilitate correct protein folding and easier downstream processing (29). Thus, the direct immobilization and easy purification of the enzyme reported here were considered as industrially advantageous.…”

Section: Discussionmentioning

confidence: 98%

One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli

Rasiah

Rehm

2009

Appl Environ Microbiol

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Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable ␣-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granuleforming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting ␣-amylase activity. The ␣-amylase beads were assessed with respect to ␣-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized ␣-amylase showed Michaelis-Menten enzyme kinetics exerting a V max of about 506 mU/mg of bead protein with a K m of about 5 M, consistent with that of free ␣-amylase. The stability of the enzyme at 85°C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, costeffective method for the production of immobilized ␣-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.␣-Amylases (EC 3.2.1.1) are among the most widely used technological enzymes, with applications in food processing, detergent manufacture, and several other industries (5, 13, 25) that use starch liquefaction (21). While they are widely distributed in plants, animals, and microorganisms, the secreted ␣-amylase from Bacillus licheniformis (BLA) is the one most extensively used in the starch industry (5), due mainly to its thermostability (7, 13). However, BLA has been subjected to protein engineering approaches in order to enhance its technically relevant enzyme properties such as greater temperature stability and a wider pH activity range (16,24).Industrial enzymes are frequently immobilized onto solid supports in order to increase resistance to fluctuations in conditions such as pH and temperature (18) and to facilitate repeated usage. Although BLA has previously been successfully immobilized (25,27,28), it has been suggested that access to large starch molecules through pores in typical supports can be a limitation (27). Therefore, a system that allows free, high-density contact between the immobilized enzyme and its substrate would be an advantage. However, covalent immobilization of BLA as well as any other biocatalyst requires a laborious and costly production process involving production of the enzyme, generation of supports, and chemical crosslinking of both components. ...

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“…Among bacteria, species of genus Bacillus are widely used for the α-amylase production. Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquefaciens are known to be good sources of α-amylase and have been used for commercial production of the enzyme for various industrial applications (Shahhoseini et al 2003;Gangadharan et al 2006Gangadharan et al , 2008Sivaramakrishnan et al 2006). Due to industrial importance, several enzymes from the α-amylase family were subjected to overproduction in different recombinant systems (Sibakov 1986;Suominen et al 1995;Marco et al 1996;Jorgensen et al 1997;Lin & Hsu 1997;Park et al 1997).…”

Section: Introductionmentioning

confidence: 99%

Insoluble but enzymatically active α-amylase from Bacillus licheniformis

et al. 2009

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Abstract:The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.

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Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene

Cited by 25 publications

References 18 publications

Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli

Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli

One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli

Insoluble but enzymatically active α-amylase from Bacillus licheniformis

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